The small soil amoeba, Acanthamoeba castellanii, is being used as a model system to study the flow of membrane through successive cellular compartments during endocytosis. We have developed greatly improved preparative procedures for ultrastructural studies of the membrane systems in this amoeba. Rapid freezing in -190 degrees propane, followed by freeze substitution, adequately preserves for the first time a complex system of vesicles and tubules that function in membrane and content transport. Rapid freezing also stabilizes the forming fluid-uptake vesicles and shows that fluid uptake vesicles are non-coated and have the same mechanism of release into the cytoplasm as phagosomes. The neck of the endosome elongates and is constricted by microfilaments, releasing the endosome to move in the cytoplasmic flow. In order to follow the path of membrane that enters the cell during endocytosis, we have used a gold-labeled monoclonal antibody that is specific for membrane proteins. Within 1-2 minutes after the cell surface was labeled with the specific gold probe, the label was found in small vesicles and tubules. Within 10 minutes the label appeared in large (diameter >1 (mu)m) vacuoles. The large vacuoles contain acid hydrolases and are a degradative compartment. The gold label was also present at the earliest time intervals in intermediate size vesicles that contained membrane remnants, suggesting that a considerable fraction of membrane is degraded immediately after internalization. Thus the small vesicle compartment may be a sorting compartment analogous to the early endosomes in mammalian cells.